GC-MS Analysis, Heavy Metals, Biological, and Toxicological Evaluation of Reseda muricata and Marrubium vulgare Methanol Extracts

The usage of herbal remedy is growing vividly all around the world. Though, ecological contamination particularly with heavy metals carriages thoughtful problem on quality of medicinal plants and their foodstuffs. In the world, 80% of the population depend on traditional medicine, while information on the levels of heavy metal such as Zn, Mn, Cu, Cr, Pb, As, Cd, and Cr in plants utilized for making of herbal remedies is unavailable. Therefore, the purpose of this study was to assess phytochemicals, biological activities, and heavy metal analysis of Reseda muricata and Marrubium vulgare grown in different parts of Saudi Arabia. Qualitative phytochemical analysis of R. muricata and M. vulgare confirmed the presence of alkaloids, flavonoids, tannins, phenol, and saponins. Methanol extracts of both Reseda muricata and Marrubium vulgare were characterized with the help of GC-MS. Antioxidants, antimicrobial, and brine sharp lethal toxicity of the both species were also evaluated.


Introduction
e medicinal potential of plants species is because of the occurrence of secondary phytoconstituents which have numerous functions such as antioxidant, antimicrobial, cytotoxic, anticancer, and antiviral. Plants have been the main root of traditional medicines since ancient times [1][2][3]. e plants are used as primary healthcare all over the world, but South American countries in particular [4]. About 300-315 thousand species of plants are present on this planet, and few of these provide food to humans, aquatic, and terrestrial animals [5]. Plants are used as antimicrobial agents that are used to kill microorganisms such as bacteria and fungi or inhibit their growth [6,7]. ey also have antioxidant, anticancer, antiviral, and cytotoxic and much more effects. Antioxidants inhibit the oxidation of substrate such as free radicals [8,9]. Cytotoxicity is the quality of being toxic to cells. Cytotoxic potential of crude extract is ascertained against various cells using methods such as MTT and brine shrimp lethality assays [10,11]. Reseda muricata is an herb belonging to family and spread in southeast Egypt. Family Resedaceae consists of 6 genera and 75 species. While, very small data about R. muricata appeared till date. Previously, chemical screening of R. muricata has reported a flavonoid trioside, and its coumaryl ester together with some flavonoids and phenolic acids were identified from the leaves of Reseda muricata. Pharmacological studies of extracts of various Reseda species presented antifungal, antibacterial, and anti-inflammatory activities. R. muricata is traditionally used for the treatment of hemorrhoids, stomach aches, and diarrhea [12].
Marrubium vulgare is a plant of family Lamiaceae, whose genus comprises of 97 species. It is spread far and wide along the Mediterranean Sea and growing in the temperate areas of the Eurasian region. e plant has been utilized as a substitute for hops in beer breweries and is presently used to make herbal teas. In the past times, it was augmented to boiled vegetables, sauces, and salads. It is also utilized as traditional medicine. M. vulgare is habitually a significant foundation for the pharmaceutical and food industries. For instance, just in India, there are 33 registered herbal formulations comprising of white horehound. M. vulgare showed diaphoretic, expectorant, stimulant, tonic, diuretic, and aromatic potential. Its phytochemical constituents are diterpenic lactones, phenolic compounds, and phenylpropanoids [13]. Folk usage of Reseda muricata fruit is reported as menstruation tonic [14]. Similarly, in Morocco, the leaf infusion utilization of M. vulgare is used for the treatment of metabolic disorder [15]. Furthermore, in Serbian language, M. vulgare is called očajnica, which means desperate woman; because its tea is considered good remedy for women who were unable to conceive and regulate menstrual cycle, it is also traditionally utilized for the treatment of respiratory and gastrointestinal disorders [16]. Medicinal uses of the plants are due to the presence of phytochemicals. e most generally responsible phytoconstituents as antidiabetic are benzoic acid derivatives, phenol and its derivatives, flavonoid, amino acid and its derivatives, vitamins, saponin, alkaloid, and carbohydrate [17]. Phytochemicals such as flavonoids, polyphenols, steroids, terpenoids, and alkaloids have balanced usages and are existed in different amounts in different plants species. e presence of these classes of phytochemicals in plants species and dietary food plays a substantial part to defend against ailment [18]. Keeping in mind the importance of these plants species, we have designed the current study to evaluate its biological potential. e plants R. muricata and M. vulgare parts were ground to fine powder by a mechanical grinder. e powder plant material (100 g, each) was soaked in 3 liters of methanol. e greenish methanol was filtered and evaporated with the help of a rotary evaporator. e greenish residue of methanol was further assessed for phytochemicals screening, antimicrobial, antioxidant, and cytotoxic potential using reported protocols.

GC-MS Analysis.
Agilent GC 7890A combined with a triple axis detector 5975 C single quadrupole mass spectrometer were used for GC-MS analysis. e chromatographic column was an Agilent HP 5MS column (30 m × 0.25 mm × 0.25 µm film thickness), with highpurity helium as the gas carrier, at a flow rate of 1 mL/ min. e injector temperature was 250°C, and it was equipped with a splitless injector at 20 : 1. e source temperature of MS was set at 230°C, and the quad temperature was set at 150°C e oven temperature was initially at 40°C (held for 1 min), then was increased to 150°C at 10°C min −1 (held for 1 min), and then increased further to 300°C at 10°C min −1 for 1 min. e injection volume was 1 μL, and the scan range was set at 50-800 mass ranges at 70 eV electron energy and the solvent delay of 3 minutes. Finally, unknown compounds were identified by comparing the spectra with that of the NIST 2008 (National Institute of Standard and Technology library). e total time required for analyzing a single sample was 29 minutes.

Agar Well Diffusion Assay (Antibacterial Activity).
Methanol extracts of the plants R. muricata and M. vulgare were evaluated for antibacterial potential using the well diffusion assay. Nutrient agar media plates were inoculated and were placed in the incubator at 37°C for 18-24 h. Wells were designed in Petri dish having 6 mm diameter with the help of sterile cork borers. Using a sterile swab, the inocula of the respective bacterial strains were spread on nutrient agar plates and then dried at 37°C for 15 min. Stock solutions of n-hexane, chloroform, ethyl acetate, and butanol extracts were prepared using DMSO as solvent. Concentration of each extract solution was kept as 2 mg/mL and 3 mg/mL. en, 100 μL of each extract was administered in each well containing E. coli, K. pneumonia, Xanthomonas, and S. aureus.
e Petri plates were placed for incubation at temperature 37°C for 24 h. e zone of inhibition in (mm) for antibacterial activity was determined after incubation. e positive control used showed antibacterial activity [25,26].

Agar Well Diffusion Assay (Antifungal Activity).
Agar well diffusion assay was used to find out the antifungal activity of R. muricata and M. vulgare. e fungal strains were first grown on Petri plates. Wells of 6 mm in diameter were made in Petri plates containing nutrient agar medium using sterile cork borers. Using a sterile swab, the inocula of the respective bacterial strains were spread on nutrient agar plates and then dried at 37°C for 15 min. Stock solutions of n-hexane, chloroform, ethyl acetate, and butanol extracts were prepared using DMSO as solvent. Concentration of each extract solution was kept as 2 mg/mL and 3 mg/mL. en, 100 μL of each extract was administered in each well containing Aspergillus niger, clinical Candida, Acremonium, Rhizopus, and Trichoderma. e Petri plates were placed for incubation at temperature 37°C for 24 h. e zones of inhibition in mm for antibacterial activity were determined after incubation. e positive control used showed antibacterial activity [27,28]. Evidence-Based Complementary and Alternative Medicine

Antioxidant Activity.
e antioxidant activity of R. muricata and M. vulgare was measured by using 2,2diphenyl picrylhydrazyl radical (DPPH). e molecule responsible for antioxidant activity reacts with DPPH and converts it into diphenyl-picryl hydrazine having yellow color. is change in color is measured with the help of a spectrophotometer. Using the following equation, the DPPH radical inhibiting activity or antioxidant activity of the plant extract with different solvents was calculated.
where Abs (control) and Abs (test) are the absorbance of the control and absorbance of the tested sample, respectively [29].

Brine Shrimp Lethality
Protocol. e cytotoxic activity of the plant was assessed using the brine shrimp lethality bioassay method where totally 6 graded doses (3 graded doses for each plant) (1000 μg/mL, 100 μg/mL, and 10 μg/ mL) were used. Brine shrimps (Artemia salina Leach) nauplii (Ocean 90, USA) were used as test organisms. For hatching, eggs were kept in brine with a constant oxygen supply for 48 h. e mature nauplii were then used in the experiment. DMSO was used as a solvent and also as a negative control. Vincristine sulfate was used as a reference standard in this case. To gage the percentage mortality (% M), the number of dead shrimps is divided by the total number of shrimps and is then multiplied by 100%. e death of the shrimps confirmed the presence of bioactive compounds in the plant extracts [21,28,30].

Determination of Heavy Metals.
e protocol described by W. Khan et al. [31] was used. Analytical grade concentrated per chloric acid (HClO 4 ) and nitric acid (HNO 3 ) were used for the digestion. Samples (0.2 g) of dry grounded whole plant (each) were weighed into 100 mL beakers. Predigestion of the samples was performed with HNO 3 (5 mL), followed by cooling and digestion again to HC1O 4 fumes. Heavy metals analyses for zinc (Zn), copper (Cu), manganese (Mn), chromium (Cr), lead (Pb), arsenic (As), cadmium (Cd), and chromium (Cr) were performed in triplicates for both medicinal plants using the atomic absorption spectrophotometer (Analyst 700, Perkin Elmer).

GC-MS Analysis of Reseda muricata (RMM) and Marrubium vulgare (MVM) Methanol Extracts. GC-MS analysis
of Reseda muricata and Marrubium vulgare methanol extracts is shown in Figure 1, Table 1 and Figure 2, Table 2, respectively. GC-MS analysis of RMM showed 17 constituents in methanol extracts. e highest abundance of 2propenenitrile, 3-phenyl-, (E)-followed by 1H-indole-3acetic acid-methyl ester has been observed as shown in Figure 1. Similarly, 42 phytoconstituents were noted in the methanol extract of MVM. 5-Methoxy-2-nitrobenzoic acid was the highest in abundance followed by hexadecanoic acid and methyl ester in MVM. Phytochemicals as collective in the extracts are responsible for biological activities of plant extracts. erefore, detail of phytochemicals is important.

Qualitative Phytochemicals
Analysis of R. muricata and M. vulgare. Qualitative investigation of methanol extract of R. muricata and M. vulgare confirmed the presence of different kinds of phytochemicals such flavonoids, phenols, saponins, alkaloids, and tannins, as given in Table 3.

Antibacterial Activity.
e bacterial strains used are E. coli, Klebsiella pneumonia, Xanthomonas, and Staphylococcus aureus.
e methanol extract of R. muricata and M. vulgare showed antibacterial activity (Table 4). Both the extracts showed a significant zone of inhibition against the test bacterial strain, which confirmed that the methanol extract of R. muricata and M. vulgare can be used for further investigation.

Antifungal Activity.
A. niger, Candida, Rhizopus, Acremonium, and Trichoderma fungal stains were used. e results obtained are given in Table 5. e methanol extract of R. muricata and M. vulgare showed significant antifungal activities.

Antioxidant Activity.
e DPPH free radical scavenging assay was used to evaluate the antioxidant potential of the methanol extract of R. muricata and M. vulgare which showed that both extracts are active (Table 6). e results were compared with ascorbic acid which was used as a standard antioxidant having LC 50 31.59 μg/mL. e LC 50 of both extracts was above 100 μg/mL (Table 6).
3.6. Cytotoxicity. Brine shrimp lethality assay (BSLA) was applied to evaluate the cytotoxicity of the extracts of Reseda muricata. It can be seen that with increased in concentration, the mortality rate was increased. e extracts were found to have high mortality above 1000 μg/mL mortality rate and LD 50 , as given in Table 7.

Determination of Heavy Metals.
e results given in Table 8 provide the levels of heavy metal in the whole part of R. muricata and M. vulgare.
ese results show that the concentration of bioessential elements is within the permissible limit as well as the toxic heavy metal not detected in it.
According to the FAO/WHO [31], the acceptable limit for Zn, Cu, Cr, and Mn is 27.4, 3.00, 0.02, and 2 ppm, respectively. From the given findings, it is concluded that the levels of these elements are within the standard parameters. Our study completely agreed with the results which confirmed that concentration of heavy metals in both species R. muricata and M. vulgar were within the toxic limits.
ough, its concentration varies based on different Evidence-Based Complementary and Alternative Medicine environmental conditions [32]. ese metals are very necessary for the development of plants, and its excess or shortage can cause serious problems in plants and humans. Zn is an essential trace nutrient for plant growth due to its role in several cell functions. It is also vital for brain growth, normal growth, bone formation, wound-healing, and behavioral response with a dietary limit in humans of 100 ppm [33]. Its deficiency causes diabetes and loss of smell and touch. Cu is important for normal plant growth, but its extreme levels (>100 ppm) can cause phytotoxicity. e concentration of Cu in seed and flower parts exceeded the prescribed WHO limits, but its concentration was lower in leaf, stem, and root. Meanwhile, with respect to bodyweight, the acceptable lower limit of Cu is 20 μg/mg bodyweight per day [31,34]. In both R. muricata and M. vulgare samples, Pb, As, Cd, and Cr were not detected. Industries, sewage, air, water pollution, and the fly ash are the main sources of heavy metal contamination. ese plants were collected from the Hawdaf Sudayeer Dam of Saudi Arabia, the dam constructed only to store rain water. It is away from cities and industries or industries sewage.
is may be the reason why these plants are safe. A similar study reports that three other           [36]. e poisonous effects of these heavy metal are because of their hindrance with the regular body biochemistry in normal metabolic processes. Usually, Cr and arsenic are the heavy metals most often concerned in morbidity and death [37]. Likewise, the plant metabolites are fundamental phytoconstituents that display a few observable impacts in the human body. Flavonoids and alkaloids possess strong antioxidant, anticancer, and antimalarial activities. Steroids are very vital class of alcohols with variable implication. e structures are so appropriate to be efficiently transformed by the microbial activities to commercially valued constituents; otherwise, it is hard to synthesize [38]. e presence of alkaloid and tannin contents in R. muricata was also quantitively and qualitatively confirmed by another study [39]. ough, till date, very fewer biological investigations have been made with R. muricata, Reseda species have been described to hold numerous biological potential such as antimicrobial, antioxidant, and anti-inflammatory, which agree with our findings. e presence of steroids in all extracts modeled a limitless anticipation.
e saponins are also identified as the soap forming constituents and are commercially significant [40]. e extracts displayed a good variety of dose-reliant antimicrobial actions against the tested pathogenic fungi and bacteria. So, this plant may be useful for the preparation of broad-spectrum antibacterial drugs. To summarize, this study is a decent help for diverse folklore usages of plants, but still needed a broad study and care because different geographical conditioned may affect the heavy metal contents as well as phytochemical parameters.

Conclusion
Traditionally, both plant species have many biological potentials. Numerous phytoconstituents, counting tannins, steroids, alkaloids, phenol, saponin, and flavonoid, are present in methanol extract of R. muricata and M. vulgare. e presence of heavy metals such as Zn, Cu, Cr, and Mn are in permissible limits. e paper disc diffusion test displayed some significant antimicrobial effectiveness compared to the standard antibiotics. Antioxidant effectiveness possessed by a plant is certain to keep the free radicals in the body and scavenge them if they are high and a very excellent protector to DNA. Hence, this study recommended further investigation to isolate new bioactive compounds from R. muricata and M. vulgare.

Data Availability
e data used to support this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.